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Molecular Endocrinology 11 (10): 1552-1562
Copyright © 1997 by The Endocrine Society

Phosphoinositide 3-Kinase Is Required for Insulin-Induced but Not for Growth Hormone- or Hyperosmolarity-Induced Glucose Uptake in 3T3-L1 Adipocytes

Hiroshi Sakaue, Wataru Ogawa, Masafumi Takata, Shoji Kuroda, Ko Kotani, Michihiro Matsumoto, Motoyoshi Sakaue, Shoko Nishio, Hikaru Ueno and Masato Kasuga

Second Department of Internal Medicine (H.S., W.O., M.T., S.K., K.K., M.M., M.S., M.K.), Kobe University School of Medicine, Kobe 650, Japan,
Molecular Cardiology Unit (S.N., H.U.), Kyushu University School of Medicine, Fukuoka 812-82, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The regulatory mechanism of glucose uptake in 3T3-L1 adipocytes was investigated with the use of recombinant adenovirus vectors encoding various dominant negative proteins. Infection with a virus encoding a mutant regulatory subunit of phosphoinositide (PI) 3-kinase that does not bind the 110-kDa catalytic subunit ({Delta}p85) inhibited the insulin-induced increase in PI 3-kinase activity coprecipitated by antibodies to phosphotyrosine and glucose uptake in a virus dose-dependent manner. Overexpression of a dominant negative RAS mutant in which Asp57 is replaced with tyrosine (RAS57Y) or of a dominant negative SOS mutant that lacks guanine nucleotide exchange activity ({Delta}SOS) abolished the insulin-induced increase in mitogen-activated protein kinase activity, but had no effect on PI 3-kinase activity or glucose uptake. Although GH and hyperosmolarity attributable to 300 mM sorbitol each promoted glucose uptake and translocation of glucose transporter (GLUT)4 to an extent comparable to that of insulin, these stimuli triggered little or no association of PI 3-kinase activity with tyrosine-phosphorylated proteins. Overexpression of {Delta}p85 or treatment of cells with wortmannin, an inhibitor of PI 3-kinase activity, had no effect on glucose uptake or translocation of GLUT4 stimulated by GH or hyperosmolarity. Moreover, overexpression of {Delta}SOS or RAC17N also did not affect the increase in glucose uptake induced by these stimuli. A serine/threonine kinase Akt, a constitutively active mutant of which was previously shown to stimulate glucose uptake, is activated by insulin, GH, and hyperosmolarity to ~4-fold, ~2.1-fold, and ~2.3-fold over basal level, respectively. These results suggest that insulin-induced but neither GH- or hyperosmolarity-induced glucose uptake is PI 3-kinase-dependent, and neither RAS nor RAC is required for glucose uptake induced by these stimuli in 3T3-L1 adipocytes.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Stimulation of glucose uptake is one of the most important short-term actions of insulin. Although the effects of insulin on various biological activities are mediated by activation of the insulin receptor tyrosine kinase (1), the mechanism by which receptor activation increases glucose uptake is not fully understood. Phosphoinositide (PI) 3-kinase is thought to play a pivotal role in this process. This lipid kinase consists of a 110-kDa catalytic subunit (p110) and an 85-kDa regulatory subunit (p85) that contains SRC homology 2 and 3 domains (2, 3). In response to insulin, insulin receptor substrate (IRS)-1, the best characterized substrate of the insulin receptor kinase, undergoes tyrosine phosphorylation and binds to p85 through its SRC homology 2 domains (3), an interaction that is thought to be crucial for activation of the enzyme (2, 3).

The importance of PI 3-kinase in glucose uptake was revealed with the use of a dominant negative mutant of p85 (4) and wortmannin, a fungal metabolite that inhibits the catalytic activity of p110 at nanomolar concentrations (5). Wortmannin prevents the insulin-induced increase in glucose uptake in various cells and tissues (3, 5). However, wortmannin inhibits not only the catalytic subunit of PI 3-kinase but also mammalian Vps34 homolog (also known as phosphatidylinositol-specific PI 3-kinase) (6), p110{gamma} (also known as G protein ß{gamma} subunit-activated PI 3-kinase) (7), a novel PI 4-kinase (8), phospholipase A2 (9), and mTOR/FRAP, a putative target of rapamycin (10), indicating that wortmannin is not a specific inhibitor of PI 3-kinase. Although the synthetic agent LY294002 inhibits the catalytic activity of PI 3-kinase and insulin-induced glucose uptake at concentrations 100 times those of wortmannin (5, 11), it is not known whether PI 3-kinase is the only target of this reagent.

When overexpressed in cultured cells, a mutant p85, which lacks the binding site for p110, inhibited the association of endogenous PI 3-kinase with IRS-1 (4). In these cells, both insulin-induced glucose uptake and the translocation of glucose transporters (GLUTs), which is thought to be essential for glucose uptake (12), were markedly attenuated (4), indicating that the association of the lipid kinase with IRS-1 is required for this process. Although overexpression of the mutant p85, which we have termed {Delta}p85, might be expected to inhibit PI 3-kinase activity more specifically than pharmacological approaches, the effect of this protein on glucose uptake has been examined only in Chinese hamster ovary (CHO) cells, a fibroblast cell line. Fibroblasts express GLUT1 glucose transporters, which are less sensitive to insulin than the GLUT4 glucose transporters that are expressed by the physiological target cells of insulin (12). Furthermore, these two glucose transporters reside in different vesicles (13). The regulation of glucose transport in fibroblasts may thus differ from that in adipocytes or muscle cells. Although we have shown that microinjection of {Delta}p85 or transient transfection with {Delta}p85 cDNA inhibits GLUT4 translocation in adipocytes (14, 15), it is not known whether the association of PI 3-kinase with IRS-1 is required for glucose uptake in the physiological target tissues of insulin.

Whether RAS and the signaling pathways initiated by this GTPase plays a role in insulin regulation of glucose uptake is controversial. Introduction of a constitutively active RAS was shown to promote translocation of GLUT4 (12, 16), an effect that might be mediated by PI 3-kinase because activated RAS stimulates PI 3-kinase activity in intact cells (17). On the other hand, inhibition of RAS activation by a dominant negative mutant had no effect on insulin-induced translocation of GLUT4 or glucose transport in adipocytes (18, 19). Another complicating factor in evaluating the role of RAS-mitogen-activated protein (MAP) kinase pathway in glucose uptake is that insulin-induced MAP kinase activity is inhibited by wortmannin (20), suggesting that the effect of wortmannin on glucose uptake may be partly mediated by the RAS-MAP kinase pathway.

In addition to insulin, a variety of extracellular stimuli, including GH, hydrogen peroxide, vanadium compounds, and inhibitors of serine or threonine phosphatases such as okadaic acid (21, 22, 23, 24), stimulates glucose uptake or GLUT4 translocation. Furthermore, cellular stresses such as hypoxia, increase in heat, pH, hyperosmolarity, or contraction (25, 26, 27, 28) also stimulate glucose uptake. Although it is less well characterized than insulin-induced glucose uptake, the molecular mechanism of insulin-independent glucose uptake is also of interest because muscle contraction, which promotes glucose uptake in the absence of insulin (27, 28), is thought to be important in reducing plasma glucose concentrations in individuals with diabetes.

With the use of recombinant adenovirus vectors to introduce dominant negative molecules into differentiated mouse 3T3-L1 adipocytes, we have now investigated the roles of PI 3-kinase, RAS, and RAC in regulating glucose transport induced by GH, hyperosmolarity, and insulin.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Transient expression of dominant negative proteins in cultured cells is widely used to block a specific signaling pathway. Although 3T3-L1 adipocytes are a good model with which to study the mechanism of glucose transport because of their high responsiveness to insulin, an efficient method for achieving transient gene expression in these cells has not been established. To introduce exogenous genes into the cultured adipocytes, we used the replication-defective recombinant adenovirus vector, Adex (29). We first tested the efficiency of gene transfer with the use of Adex encoding ß-galactosidase (AdexCALacZ). Six to 10 days after differentiation, the adipocytes were infected with AdexCALacZ at various multiplicity of infections (MOIs). After 48 h, the cells were subjected to ß-galactosidase staining or immunoblot analysis with antibodies to ß-galactosidase. Cytochemistry revealed that the extent of ß-galactosidase expression increased in an MOI-dependent manner, so that >95% of cells were positive for staining at an MOI of 30 (Fig. 1Go, A and B). Immunoblot analysis also revealed the MOI-dependent increase in ß-galactosidase expression (Fig. 1CGo). These results demonstrated a high efficiency of gene transfer with Adex.



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  		Figure 1. Expression of ß-Galactosidase from the AdexCALacZ Vector in 3T3-L1 Adipocytes

Cells were infected with AdexCALacZ at the indicated MOIs and, after 48 h, subjected to ß-galactosidase staining [macroscopic view (A), x400 magnification (B)] or immunoblot analysis with antibodies to ß-galactosidase (C). One-twentieth of the lysate from a 6-cm plate was applied to each lane in panel C.

 
We therefore prepared Adexes that encode a mutant regulatory subunit of PI 3-kinase that lacks the binding site for the 110-kDa catalytic subunit ({Delta}p85), a mutant RAS in which Asp57 is replaced by tyrosine (RAS57Y), a mutant SOS that lacks guanine nucleotide exchange activity ({Delta}SOS), and a mutant RAC in which Ser17 is replaced by asparagine (RAC17N) and that exhibits an affinity for GDP 100 times that for GTP (30). These viruses were termed AdexCA{Delta}p85, AdexCAHRAS57Y, AdexCA{Delta}SOS, and AdexCARAC17N, respectively.

We first evaluated whether these dominant negative molecules block specific signaling pathways in 3T3-L1 adipocytes. When 3T3-L1 adipocytes were infected with various Adexes and subjected to immunoblot analyses after 48 h, all the introduced genes were expressed efficiently in an MOI-dependent manner (Fig. 2AGo, Fig. 3AGo). Infection with AdexCA{Delta}p85 inhibited insulin-induced PI 3-kinase activity that was precipitated with antibodies to phosphotyrosine in an MOI-dependent manner, with almost 95% inhibition apparent at an MOI of 30 (Fig. 2BGo). Essentially the same results were obtained when PI 3-kinase activity was precipitated with antibodies to IRS-1 (data not shown). The ability of {Delta}p85 to block the PI 3-kinase pathway was confirmed by investigating the effect of {Delta}p85 on insulin-induced glucose uptake. Infection with AdexCA{Delta}p85 inhibited insulin-induced glucose uptake in an MOI-dependent manner, with inhibition of almost 60% apparent at an MOI of 30 (Fig. 4Go). AdexCA{Delta}SOS, AdexCAHRAS57Y, and AdexCARAC17N did not affect insulin-induced PI 3-kinase activity (Fig. 3BGo) or glucose uptake (Fig. 5EGo and data not shown), indicating that the suppression of glucose uptake by infection with AdexCA{Delta}p85 was not due to nonspecific effects of virus infection but to specific inhibition of the PI 3-kinase path-way. On the other hand, overexpression of {Delta}SOS, which we have previously shown inhibits the acti-vation of RAS by insulin in CHO cells (31), as well as overexpression of RAS57Y, attenuated insulin-induced MAP kinase activity in 3T3-L1 adipocytes (Fig. 3CGo), whereas infection with AdexCA{Delta}p85 or AdexCARAC17N did not affect MAP kinase activity (Figs. 2CGo and 3BGo). Furthermore, infection of 3T3-L1 adipocytes with AdexCARAC17N, at an MOI of 30, completely inhibited insulin-induced membrane ruffling (data not shown), which is known to be regulated by RAC (30). These observations indicate that all the introduced genes with the use of Adex were capable of inhibiting specific signaling pathways in differentiated adipocytes as expected.



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  		Figure 2. Effects of Overexpression of {Delta}p85 on Insulin-Induced Activation of PI 3-Kinase and MAP Kinase in 3T3-L1 Adipocytes

A, Expression of {Delta}p85. 3T3-L1 adipocytes were infected with AdexCA{Delta}p85 at the indicated MOIs and, after 48 h, lysed. Total lysates were subjected to immunoblot analysis with antibodies to p85. One-twentieth of the lysate from a 6-cm plate was applied to each lane. B, PI 3-kinase assay. The virus-infected cells were incubated in the absence or presence of 0.1 µM insulin for 5 min, after which PI 3-kinase activity was precipitated with antibodies to phosphotyrosine and assayed. The origin and position of PI 3-phosphate (PI3P) are indicated. Data are representative of three independent experiments. C, MAP kinase assay. The virus-infected cells were incubated in the absence or presence of 0.1 µM insulin for 10 min, after which lysates were prepared and subjected to immunoprecipitation with antibodies to MAP kinase. The precipitates were then assayed for MAP kinase activity. Data are means ± SE from three experiments.

 


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  		Figure 3. Effects of Overexpression of {Delta}SOS, RAS57Y, and RAC17N on Insulin-Induced Activation of PI 3-Kinase and MAP Kinase in 3T3-L1 Adipocytes

A, Expression of {Delta}SOS, RAS57Y, and RAC17N. 3T3-L1 adipocytes were infected with AdexCA{Delta}SOS, Adex-CAHRAS57Y, or AdexCARAC17N at the indicated MOIs. Forty-eight hours after infection, the cells were lysed and the total lysates were subjected to immunoblot analysis with antibodies to SOS, to RAS, or to HA (for RAC17N). One-twentieth of the lysate from a 6-cm plate was applied to each lane. B, PI 3-kinase assay. The virus-infected cells were incubated in the absence or presence of 0.1 µM insulin for 5 min, after which PI 3-kinase activity was precipitated with antibodies to phosphotyrosine and assayed. Data are representative of three independent experiments. C, MAP kinase assay. The virus-infected cells were incubated with or without 0.1 µM insulin for 10 min, after which immunoprecipitates prepared with antibodies to MAP kinase were assayed for MAP kinase activity. Data are means ± SE from three experiments.

 


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  		Figure 4. Effects of Overexpression of {Delta}p85 on Insulin-Stimulated Glucose Uptake in 3T3-L1 Adipocytes

Cells were infected with AdexCA{Delta}p85 at the indicated MOI. Forty-eight hours after infection, the cells were treated with or without insulin, and 2-deoxy[3H]glucose uptake was assayed. Data are means ± SE from at least three experiments.

 


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  		Figure 5. Roles of PI 3-Kinase Activity and SOS in Glucose Uptake Induced by GH or Hyperosmolarity in 3T3-L1 Adipocytes

A, Effects of GH and hyperosmolarity on PI 3-kinase activity. 3T3-L1 adipocytes were incubated in the absence or presence of 0.1 µM insulin for 5 min, GH (0.5 µg/ml) for 5 min, or 300 mM sorbitol for 30 min, after which PI 3-kinase activity was precipitated with antibodies to phosphotyrosine and assayed. B, Time course of GH-stimulated PI 3-kinase activity. 3T3-L1 adipocytes were incubated with GH (0.5 µg/ml) for the indicated times, after which PI 3-kinase activity was assayed in immunoprecipitates prepared with antibodies to phosphotyrosine. Data in panels A and B are representative of three independent experiments. C and D, Effects of {Delta}p85 (C) and wortmannin (D) on glucose uptake stimulated by insulin, GH, or hyperosmolarity. 3T3-L1 adipocytes were infected with or without AdexCA{Delta}p85 (C) or preincubated in the absence or presence of 100 nM wortmannin for 20 min. The cells were subsequently exposed to insulin, GH, or sorbitol at the above concentrations and 2-deoxy[3H]glucose uptake was assayed as described in Materials and Methods. Data are means ± SE from at least three experiments. E, Effects of {Delta}SOS or RAC17N on glucose uptake stimulated by insulin, GH, or hyperosmolarity. 3T3-L1 adipocytes were infected with either AdexCA{Delta}SOS or AdexCARAC17N at an MOI of 30. Forty-eight hours after infection, the cells were treated with insulin, GH, or sorbitol, and 2-deoxy[3H]glucose uptake was assayed. Data are means ± SE from at least three independent experiments.

 
With the use of the various mutant proteins described above, we investigated the molecular mechanisms of glucose uptake induced by GH and sorbitol hyperosmolarity, both of which are known to stimulate glucose uptake, and compared them with that by insulin. When 3T3-L1 cells were treated with GH or sorbitol, glucose uptake was stimulated 5- to 6-fold, similar to the effect of insulin (Fig. 5Go, C–E). Maximal insulin stimulation with either GH or sorbitol did not result in further increase in glucose uptake (insulin alone, 32,575 ± 2,944 cpm; insulin plus GH, 30,725 ± 1,504 cpm; insulin plus hyperosmolarity, 28,678 ± 2,342 cpm; data are means ± SE from three experiments). Instead, glucose uptake achieved by insulin plus either GH or sorbitol was slightly decreased as compared with that achieved by insulin alone. Similar reduction was previously observed with isolated adipocytes treated with insulin plus sorbitol (26).

Sorbitol had no effect on PI 3-kinase activity precipitated with antibodies to phosphotyrosine (Fig. 5AGo). GH, which induces tyrosine phosphorylation of IRS-1 and the association of PI 3-kinase with IRS-1 in isolated adipocytes (32) and 3T3-F442 adipocytes (33), the latter of which are closely related to 3T3-L1 adipocytes, stimulated PI 3-kinase activity in a time-dependent manner (Fig. 5BGo). However, the extent of PI 3-kinase activation induced by GH was <10% of that achieved with insulin (Fig. 5AGo), whereas the extent of GH-induced glucose uptake was similar to that induced by insulin. These data suggest that the association of PI 3-kinase with tyrosine-phosphorylated protein is not required for glucose uptake induced by GH or hyperosmolarity.

To test this hypothesis, we examined the effect of overexpression of {Delta}p85 on glucose uptake induced by these stimuli. Infection with AdexCA{Delta}p85 at an MOI of 30, at which concentration the virus inhibited insulin-induced glucose uptake by ~60%, had no effect on glucose uptake induced by GH or sorbitol (Fig. 5CGo). Pretreatment of uninfected cells with wortmannin at a concentration of 100 nM abolished insulin-induced glucose uptake but had no effect on glucose uptake induced by GH or hyperosmolarity (Fig. 5DGo). Because translocation of GLUT4 is an essential step to promote glucose uptake in adipocytes, we next investigated the effect of {Delta}p85 on translocation of GLUT4 by photoaffinity labeling of membrane surface GLUT4 with the use of 2-N-4-(1-azi-2, 2, 2-trifluoroethyl)benz-oyl-1,3-bis-(D-mannos-4-yloxy)-2-propylamine (ATB-BMPA) (28, 34). Insulin as well as GH and hyperosmolarity treatment dramatically induced translocation of GLUT4 to the plasma membrane in 3T3-L1 adipocytes (Fig. 6Go). Overexpression of {Delta}p85 inhibited GLUT4 translocation induced by insulin by ~60% whereas translocation of GLUT4 promoted by neither sorbitol nor GH was affected by {Delta}p85 (Fig. 6Go), consistent with the data of glucose uptake. Involvement of PI 3-kinase to translocation of GLUT 4 by various stimuli was also examined with plasma membrane lawn assay. Insulin, GH, and sorbitol treatment provoked an increase in immunoreactivity of GLUT4 in the plasma membrane lawn prepared from 3T3-L1 adipoicytes (Fig. 7Go, B, D, and E). Pretreatment of cells with wortmannin inhibited the increase in the immunoreactivity induced by insulin (Fig. 7CGo), whereas the increase in the immunoreactivity promoted by either GH or hyperosmolarity was not affected by wortmannin (Fig. 7Go, E and G). These observations confirmed that PI 3-kinase is not required for glucose uptake stimulated by GH or sorbitol.



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  		Figure 6. Effects of Overexpression of {Delta}p85 on Translocation of GLUT4 in 3T3-L1 Adipocytes

Cells were infected with AdexCA{Delta}p85 at the indicated MOI. Forty-eight hours after infection, the cells were incubated with or without insulin (A), GH (B), or sorbitol (C). Translocation of GLUT4 to the plasma membrane was assayed by ATB-[2-3H]BMPA surface labeling as described in Materials and Methods. Data are means ± SE from at least three experiments.

 


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  		Figure 7. Effects of Wortmannin on Immunofluorescence Labeling of GLUT4 in Plasma Membrane Lawn Prepared from 3T3-L1 Adipocytes

Cells were incubated in the absence (A, B, D, and F) or presence (C, E, and G) of 100 nM wortmannin for 20 min and in the absence (A) or presence of insulin (B and C), GH (D and E), or sorbitol (F and G), after which plasma membrane fragments were prepared for immunofluorescence microscopy with antibodies to GLUT4 (1F8) and TRITC-labeled secondary antibodies. Data are representative of at least three independent experiments.

 
We also examined whether signaling pathways mediated through RAS or RAC are involved in glucose uptake stimulated by GH or hyperosmolarity. Overexpression of {Delta}SOS or RAC17N had no effect on glucose uptake induced by GH, hyperosmolarity, or insulin (Fig. 5EGo). Similar results were obtained with a virus that encodes RAS57Y (data not shown). These observations indicate that the signaling pathways initiated by RAS or RAC do not mediate the activation of glucose uptake by these stimuli in 3T3-L1 adipocytes.

Finally, we examined whether these stimuli activate serine/threonine kinase Akt, a constitutively active mutant of which has been shown to promote glucose uptake as well as translocation of GLUT4 in 3T3-L1 adipocytes (35). Insulin stimulated Akt activity to ~4-fold, and GH or sorbitol stimulated to ~2.1- and ~2.3-fold over basal level, respectively (Fig. 8Go).



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  		Figure 8. Effects of Insulin, GH, or Sorbitol on Akt Activity in 3T3-L1 Adipocytes

3T3-L1 adipocytes were treated with or without 0.1 µM insulin for 5 min, GH (0.5 µg/ml) for 5 min, or 300 mM sorbitol for 30 min, after which cells were lysed and precipitated with polyclonal antibodies against Akt. The precipitates were then assayed for Akt kinase activity. Data are means ± SE from four independent experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The 3T3-L1 cell line is a useful model for investigating the metabolic actions of insulin. Under appropriate conditions, the originally fibroblast-like cells acquire many characteristics of adipocytes, including accumulation of lipids, expression of GLUT4 and insulin receptors, and an increased sensitivity of glucose uptake to insulin (12). Many investigators have studied insulin signaling in 3T3-L1 adipocytes by introducing exogenous genes into the preadipocytes (12, 35, 36, 37). However, with such an approach, the exogenous genes may affect the adipogenesis, given that insulin is a potent adipogenic stimulus. Indeed, a recent report has shown that introduction of a constitutively active mutant of Akt, a serine/threonine kinase known to be activated by insulin, into 3T3-L1 preadipocytes accelerates differentiation and enhances expression of GLUT1 protein (35). Circumvention of this problem requires a method for gene transfer into differentiated adipocytes. Despite the fact that vaccinia virus-mediated gene transfer is effective in a certain cell lines (38), we have found that 3T3-L1 adipocytes are not a good target for the vaccinia system. We therefore attempted to introduce exogenous genes into the differentiated adipocytes with the use of replication-defective recombinant adenovirus vectors, which have proved effective for gene transfer, especially in quiescent, differentiated cells and tissues (29).

We have prepared adenovirus vectors that encode dominant negative mutants of PI 3-kinase, RAS, SOS, and RAC. We previously reported that overexpression of {Delta}p85 in fibroblasts attenuates insulin-induced glucose uptake as well as translocation of GLUT1 (4), and microinjection of {Delta}p85 inhibits insulin-induced translocation of GLUT4 in adipocytes (14). We have shown that overexpression of {Delta}p85 with the use of adenovirus vector inhibited insulin-induced increase in PI 3-kinase precipitated with antiphosphotyrosine antibodies as well as glucose uptake in 3T3-L1 adipocytes in an MOI-dependent manner. By immunoblot analysis, we compared the amount of GLUT4 and insulin receptor in adipocytes that had been infected with or without AdexCA{Delta}p85 and found that no detectable change in the amount of these proteins was detected within 48 h after infection (data not shown). {Delta}SOS or RAS57Y inhibited MAP kinase activity induced by insulin without affecting PI 3-kinase activity precipitated by antibodies to phosphotyrosine or insulin-induced glucose uptake. Furthermore, overexpression of RAC17N blocked insulin-induced membrane ruffling. All these data indicate that gene transfer with the use of Adex is a useful method to investigate signal transduction in differentiated adipocytes.

Although it has been reported that wortmannin inhibits insulin-induced activation of MAP kinase (20), overexpression of {Delta}p85 did not affect MAP kinase activity. These data may suggest that some uncharacterized wortmannin-sensitive molecule may lie upstream of MAP kinase. Recently, p110{gamma}, the catalytic activity of which is known to be inhibited by wortmannin, was shown to regulate MAP kinase activation stimulated by heterotrimeric G protein-coupled receptor (7), suggesting that p110{gamma} or a similar enzyme that can transmit signals to MAP kinase may also be involved in insulin-induced MAP kinase activation.

A number of stimuli other than insulin promote glucose uptake in various cells and tissues, including fat and muscle cells. Although some of these stimuli induce glucose uptake or GLUT4 translocation in a wortmannin-insensitive manner (25, 26, 28), it is not yet known whether GH and hyperosmolarity stimulate glucose uptake or GLUT4 translocation via a PI 3-kinase-dependent mechanism. We have now shown that, in 3T3-L1 adipocytes, GH and hyperosmolarity increased glucose transport and translocation of GLUT4 to a similar extent as insulin. The observation that these stimuli induced little or no association of PI 3-kinase with tyrosine-phosphorylated proteins, together with the failure of {Delta}p85 and wortmannin to inhibit stimulated glucose uptake and translocation of GLUT4, indicates that GH and hyperosmolarity promote glucose uptake via a pathway (or pathways) that bypasses or is completely independent of PI 3-kinase.

Maximal insulin stimulation with neither GH nor hyperosmolarity resulted in a further increase in glucose uptake. In isolated muscle, the combined effects of contraction, which stimulates glucose uptake via a wortmannin-insensitive mechanism, and maximal insulin stimulation on glucose uptake are additive (28), suggesting that signaling pathways of glucose uptake elicited by contraction in muscle and by either GH or by sorbitol in adipocytes may be different.

Although earlier reports have suggested that RAS and RAC are not involved in regulatory mechanism of glucose uptake stimulated by insulin (18, 19, 39), it has not yet been determined whether these GTP-binding proteins regulate glucose uptake elicited by other stimuli. We thus investigated the effects of {Delta}SOS and RAC17N on sorbitol- or GH-induced glucose uptake. Both sorbitol and GH, the latter of which is known to activate RAS-MAP kinase cascade (40), do not seem to require RAS activation for glucose uptake because overexpression of {Delta}SOS did not affect glucose uptake. RAC is known to regulate stress-activated protein kinase pathway, p38 MAP kinase pathway, and p21-activated protein kinases (41, 42, 43). Hyperosmolarity as well as various cellular stresses including heat shock, UV, ionizing radiation, and oxidative stress are known to activate the stress-activated protein kinase and p38 MAP kinase pathways (41). Furthermore, insulin activates p38 MAP kinase as well as p21-activated protein kinase in L6 myotube (44). We do not know whether any of these protein kinases are activated by sorbitol or GH in 3T3-L1 adipocytes, but the failure of RAC17N to block glucose uptake indicates that signals initiated by RAC activation may not be involved in glucose uptake stimulated by hyperosmolarity or GH.

Recently, Kohn et al. (35) have shown that overexpression of a membrane-targeted mutant of Akt induces glucose uptake and translocation of GLUT4 in quiescent 3T3-L1 adipocytes. Akt was originally shown to be activated by various growth factors via wortmannin-sensitive mechanisms; however, it was later found to be activated by hyperosmolarity, heat-shock (45), or ß-adrenergic agonists (46). Moreover, activation of Akt by these stimuli is not sensitive to wortmannin (45, 46). We thus investigated the effects of GH and hyperosmolarity on Akt activity in 3T3-L1 adipocytes. Although the extent of stimulation is lower than that by insulin, both GH and sorbitol activate Akt in the adipocytes. Furthermore, we have recently found that pervanadate, which is known to stimulate glucose uptake, also activates Akt in 3T3-L1 adipocytes (S. Kuroda, H. Sakaue, W. Ogawa, and M. Kasuga, unpublished observation). These data do not necessarily indicate that GH or sorbitol stimulates glucose transport through Akt activation. On the contrary, the relatively weak activation of Akt by GH or hyperosmolarity implies that signaling mechanisms in addition to Akt may be involved in glucose uptake by these stimuli. Selective inhibition of Akt activity, which might be achieved by introduction of dominant inhibitory mutants of Akt (47) by the Adex system, may be useful to elucidate a role of Akt in glucose uptake by various stimuli.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cell Culture, Antibodies, and Reagents
3T3-L1 preadipocytes maintained under an atmosphere of 7.5% CO2 in DMEM containing 25 mM glucose and supplemented with 10% heat-inactivated calf serum, 2 mM L-glutamine, penicillin (50 U/ml), and streptomycin sulfate (50 µg/ml) were induced to differentiate into adipocytes as described previously (14). Monoclonal antibodies to ß-galactosidase, to phosphotyrosine (PY20), and to the hemaggulutinin (HA) epitope tag (12CA5) were obtained from Promega (Madison, WI), Transduction Labs (Lexington, KY), and Boehringer Mannheim (Indianapolis, IN), respectively. Monoclonal antibodies to p85 (F12) and to IRS-1 (1D6) as well as polyclonal antibodies to MAP kinase ({alpha}C92), prepared against a synthetic peptide corresponding to residues 350 to 367 of rat p44MAPK, were as described previously (48, 49). Antibodies to mSOS1 (31), to H-RAS (50), to Akt (45), to GLUT4 (14), and to RAC were gifts from D. Bowtell (University of Melbourne, Melbourne, Australia), T. Tanaka (Kure Hospital, Hiroshima, Japan), U. Kikkawa (Biosignal Research Center, Kobe University, Kobe, Japan), D. James (University of Queensland, Brisbane, Australia), and Eisai Pharmaceutical Co. (Tokyo, Japan), respectively. Wortmannin (Sigma, St. Louis, MO) was dissolved in dimethyl sulfoxide, stored at -20 C in the dark, and diluted with distilled water immediately before addition to cells. Recombinant human GH was kindly provided by Novo-Nordisk (Copenhagen, Denmark).

Construction of and Infection with Adenovirus Vectors
Bovine p85{alpha} that lacks the binding site for the 110-kDa catalytic subunit of PI 3-kinase ({Delta}p85), mSOS1 that lacks the catalytic domain for guanine nucleotide exchange activity ({Delta}SOS), and H-RAS in which Asp57 is replaced by tyrosine (RAS57Y) were as described previously (4, 31, 51, 52). HA epitope-tagged RAC17N (with asparagine substituted for Ser17) was produced as described previously (53). Recombinant adenovirus vectors were generated by cloning the cDNAs into pAxCAwt (54), which contains the CAG promoter (55), and cotransfection into 293 cells with DNA-TPC, as described previously (29). Protein-encoding viruses were screened by immunoblot analysis and cloned by limiting dilution. Adenovirus vectors were propagated by a standard procedure and then purified and titrated as described previously (56). An adenovirus vector encoding RAS57Y (AdexHRAS57Y) was as described (52), and adenovirus vector encoding the lacZ gene (AdexCALacZ) (54) was a gift from I. Saito (Tokyo University, Tokyo, Japan). 3T3-L1 adipocytes were infected with Adexs at the indicated MOI (MOI = plaque-forming units per cell) in DMEM containing 10% FBS. The virus-containing medium was replaced with fresh medium after 6 h, and the cells were used for various experiments 48 h after infection.

ß-Galactosidase Staining
3T3-L1 adipocytes infected with AdexCALacZ were washed twice with PBS and fixed with 0.25% glutaraldehyde at 4 C for 10 min. After washing four times with PBS, the cells were stained with PBS containing 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (1 mg/ml), 5 mM K3Fe(CN)6, 5 mM K4(CN)6, and 2 mM MgCl2.

PI 3-Kinase Assay
3T3-L1 adipocytes were incubated in serum-free DMEM for 16 h, treated with various reagents, and then immediately frozen with liquid nitrogen. The cells were lysed in a lysis buffer described previously (38, 48), and insoluble materials were removed by centrifugation. The supernatants were subjected to immunoprecipitation with either PY20 or antibodies against IRS-1, the immunoprecipitates were washed, and PI 3-kinase activity was assayed in the immunoprecipitates as described previously (38, 48). Lysate from a 6-cm plate was used for each precipitation.

MAP Kinase and Akt Kinase Assay
MAP kinase assays (48) and Akt kinase assays (45) were performed as described with the following modifications. 3T3-L1 adipocytes were incubated in serum-free DMEM for 16 h, treated with various reagents, and then immediately frozen with liquid nitrogen. For MAP kinase assays, the cells were lysed in a solution containing 20 mM Tris-HCl (pH 7.5), 60 mM ß-glycerophosphate, 10 mM MgCl2, 0.1 mM NaF, 10 mM EGTA, 10 mM sodium pyrophosphate, 1% Nonidet P-40, 2 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 1 mM sodium orthovanadate. For Akt kinase assays, the cells were lysed as described (45). The lysates were centrifuged and the resultant supernatants were subjected to immunoprecipitation either with anti-MAP kinase antibodies, or anti-Akt kinase antibodies. After washing three times with HEPES-buffered saline (pH 7.5) containing 0.1% Triton X-100, the immunoprecipitates were incubated with 0.5 µCi of [32P]ATP in a reaction mixture containing 20 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 25 µM ATP, 1 µM PKI, and myelin basic protein (0.5 mg/ml) or Histon 2B (0.2 mg/ml) for MAP kinase assays or Akt kinase assays, respectively. After 15 min at 30 C, reactions ware terminated by the addition of SDS sample buffer. The samples were fractionated on a 15% SDS-polyacrylamide gel, and the radioactivity incorporated into either myelin basic protein or Histon 2B was determined with a Fuji BAS 2000 image analyzer (Fiji Film, Tokyo, Japan). Lysate from a 6-cm plate was used for each precipitation.

Glucose Uptake
3T3-L1 adipocytes cultured in six-well plates were incubated for 16 h in DMEM containing 5.6 mM glucose and 0.5% FBS. The cells were washed twice with DB buffer [140 mM NaCl, 2.7 mM KCl, 1 mM CaCl2, 1.5 mM KH2PO4, 8 mM Na2HPO4, (pH 7.4), 0.5 mM MgCl2] and incubated with the indicated concentrations of insulin for 15 min, GH for 10 min, or sorbitol for 60 min. One milliliter of DB buffer containing BSA (1 mg/ml) and 0.1 mM 2-deoxy-D-[1, 2-3H]glucose (1 µCi) was added to each well, and, after 5 min, the cells were washed three times with ice-cold DB buffer containing BSA (1 mg/ml) and 100 nM phloretin and then solublized with 0.1% SDS. The radioactivity incorporated into the cells was measured by liquid scintillation counting.

Photoaffinity Labeling of GLUT4 in Plasma Membrane
Photoaffinity labeling of GLUT4 was performed as described previously (28, 34) with the following modifications. 3T3-L1 adipocytes cultured in 35-mm dishes were incubated for 16 h in DMEM containing 5.6 mM glucose and 0.5% FBS. The cells were washed twice with KRH buffer (136 mM NaCl, 4.7 mM KCl, 1.25 mM CaCl2, 1.25 mM MgCl2, 10 mM HEPES, pH 7.4) and incubated with the indicated concentrations of insulin for 15 min, GH for 10 min, or sorbitol for 60 min. The buffer was removed and replaced by 200 µl of KRH buffer containing 500 µCi of ATB-[2-3H]BMPA, kindly provided by G. Holman, University of Bath, Bath, U.K.). The dishes were irradiated for 1 min using a Rayonet photochemical reactor (The Southern New England Ultraviolet Co, Branford, CT) with 300-nm lamps. The irradiated cells were washed with KRH buffer and solubilized in 1 ml of lysis buffer containing 2% nonaethyleneglycol dodecyl ether (C12E9), 5 mM sodium phosphate, 5 mM EDTA, pH 7.2, and with proteinase inhibitors aprotinin, pepstatin, leupeptin, each at 1 µg/ml. The lysates were centrifuged and the supernatants were subjected to immunoprecipitation with a monoclonal anti-GLUT4 antibody (1F8). After washing four times with PBS containing 0.2% C12E9, the labeled GLUT4 was released from the immunoprecipitates with 10% SDS, 6 M urea, and 10% mercaptoethanol and subjected to 10% SDS-PAGE. Gels were stained with Coomassie blue and sliced by lane into 8-mm slices. The slices were dried and solubilized in 30% H2O2 and 2% ammonium hydroxide, and radioactivity was quantified in a liquid scintillation counter. Labeled GLUT4 was quantified by integrating the area under the 3H peak and subtracting the average background radioactivity in the gel.

Plasma Membrane Lawn Assay and Analysis of Membrane Ruffling
GLUT4 translocation to the plasma membrane was measured by the plasma membrane lawn assay as previously described (14). In brief, 3T3-L1 adipocytes cultured on coverslips were washed in PBS and treated with 0.5 mg/ml poly L-lysine in PBS. Cells were incubated in a hypotonic buffer (1/3 x KHMgE buffer (70 mM KCl, 30 mM HEPES, 5 mM MgCl2, 3 mM EGTA, pH 7.5), and immediately broken open by placing under an ultrasonic microprobe in KHMgE buffer containing 0.1 mM phenylmethylsulfonyl fluoride and 1 mM dithiothreitol. For antibody labeling, sonicated cells were fixed in 2% paraformaldehyde, and the lawn of plasma membrane fragments was prepared with antibodies to GLUT4 (1F8) and tetramethyl rhodamine isothiocyanate (TRITC)-labeled secondary antibodies. For analysis of filamentous actin, 3T3-L1 adipocytes fixed with PBS containing 3.7% paraformaldehyde were permeabilized with 0.2% Triton X-100 for 1 min at 25 C and stained with TRITC-labeled phalloidin for 60 min at 25 C. After washing with PBS, the cells were mounted in 90% glycerol resolved in PBS containing 1 mg/ml p-phenylenediamine. Samples were examined with a fluorescence microscope (model Axiophot, Zeiss, Jena, Germany).


   ACKNOWLEDGMENTS
 
We thank Dr. J. Miyazaki for the CAG promoter; I. Saito for pAxCAwt, DNA-TPC, and technical advice on the production of adenovirus vectors; G. Holman for ATB-BMPA and technical advice on the photoaffinity labeling of GLUT4; T. Tanaka, D. Bowtell, U. Kikkawa, and D. James for antibodies.

Supported by grants from the Ministry of Education, Science, and Culture of Japan, Monbusho International Scientific Research Program, and Otsuka Pharmaceutical Co., Ltd. (to M. K.).


   FOOTNOTES
 
Address requests for reprints to: Waturu Ogawa, Second Department of Internal Medicine, Kobe University School of Medicine, 7–5-1 Kusunoki-cho, Chuo-ku, Kobe 650, Japan.

Received for publication February 18, 1997. Revision received May 28, 1997. Accepted for publication May 29, 1997.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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